Staining of D2SC1/Flt3-DC and staining of the cells to be taken up by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma
Staining of GM-DC/Raw with anti-CD11c antibodies
Coculture of DCs and cells to be taken up (1(-2)x105 + 1(-2)x105 cells in 12-well) for 4 h to 24 h at 37°C
Take supernatant and freeze at -80°C for analysis of DC-stimulation
Trypsinize cells have and fix in 4 % PFA (or formalin)
FACS analysis

We have efficiently transfected DC with RNA encoding a functional protein (E/L-Selectin) which allows entry of DC into LN from HEV.
RNA transfected human DC could be frozen and thawed without loosing their functionality.

The cells were collected and the pellet were resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) were added and the falcon were mixed by vortex. After 10’ of incubation in 65 °C water bath, the samples were incubated on ice and then centrifugated at 5000 rpm in an tabletop centrifuge.
The supernatants were transferred to a new tube and added 2 ml of acid phenol to repeat the extraction. After the centrifugation (10’ at 5000 rpm) the supernata...

Different strains of Saccharomyces cerevisiae were cultured and collected in different conditions:
* different growth phase (exponential and stationary phase)
* different growth media (standard and promoting pseudohyphal growth)
* different cell form (spheroplast, spore and whole cell)
Monocyte-derived DCs were added at a final concentration of 5x105 cells/ml into 96-well plates. Serial diluition of yeast cells and preparations were added to the MoDCs. Cytokine accumulation was evaluated in the supernatants at 24h by ELISA, according to a standard protocol and ...

Argos successfully developed a manual DC manufacturing method with excellent reproducibility in terms of quality, yield, and viability of DCs. The process developed has the following unique advantages:
- The manufacturing process has been optimized for producing DC from non-frozen day-old pheresis material which allows centralized manufacturing. The pheresis product is sent in a specially designed shipper to the manufacturing facility.
- The final vaccine product is formulated for direct injection (i.e., no clinical site participation required except for bedside thaw and i.d. injection)

We performed experiments to set up the optimal conditions for both transient transfection of synthetic siRNAs and lentiviral transduction of shRNAs into DC.
To optimize transfection efficiency, while minimizing toxicity, non targeting FITC-conjugated siRNA were transfected in two different experimental settings:
a) fresh monocytes and
b) immature DCs (iDCs) obtained from monocytes in the presence of GM-CSF/IL-4.
In both experimental settings a considerably high transfection efficiency (up to 75%) was achieved in the absence of significant cell death. Moreov...

A single step procedure for the in vitro maturation and antigen loading of human moDC was optimized. DC were electroporated with mRNA encoding tumor antigens and a putative TLR3 ligand. In vitro stimulation of naïve T cells indicated the superiority of this approach compared to in vitro stimulation with DC matured with inflammatory cytokines followed by electroporation with TAA encoding mRNA.

In 2008, much effort was spent to optimize protocols for RNA transfection in human sDCs.
- fusion proteins of the DC specific receptor DC-SIGN fused to GFP
- use of rna to express melanoma associated antigens in dendritic cells to prepare better vacines for the treatment of melanoma patients.

To determine ROS production we used the modified version by Choi et al. (2006) of the microscopic Nitroblue tetrazolium (NBT, Sigma-Aldrich) assay. Briefly, DCs were stimulated with spores or yeasts, washed, supplemented with 0.1 mg/ml of NBT, which allows the precipitation of formazan particles in presence of ROS. Blue formazan particles were dissolved using 2M KOH and DMSO and its absorbance was measured using a microplate reader at 620 nm. The absorbance of dissolved NBT increased in proportion to cell number, incubation time, and stimulus concentration.

This methodology incorporates a unique sample preparation procedure based on 96 well plate complex sample trypsinization, a nano-LC-based peptide separation coupled to MSMS analysis sequencing. This procedure led us to the identification of 600 phagosomal proteins from murine DC phagosomes.

We established protocols for the efficient in vitro transfection of primary dendritic cells based on viral and non-viral (nucleofection) methods (A. Mantei). These have been applied to modify DC surface molecules (Notch ligands) in the murine system (S. Vaddakadathou) or deliver antigens in both murine and human DC into either the MHC I or MHC II presentation pathways (A. Sattler, M. Dziubainau). For the latter we used a MHC II presentation pathway targeting vector originally published by Wang et al. 1999. Proof of principle studies for antigen delivery have been ...

Leukapheresis products from cancer patients was used as a starting material for monocyte enrichment by elutriation technology with ELUTRA® (Gambro). The monocytes were cultured for five days to immature DC (imDC´s) and subsequently transfected with different constructs encoding for the oncogene Her-2/neu and as control PSA using the technology of Amaxa biosystems or an adeno virus Her-2 full length construct. Levels of Her-2 expression was highest following transfection with 3 different truncated Transmembrane-extracellular constructs, in particular a Her-2 ra...

After 6 hour of stimulation, DCs were collected, washed 3 times with PBS, treated with zymolyase, washed twice and cells, lysated with a hypotonic solution (KCl 0.05%), were plated on YPD. Survival of yeast cells, spores or hyphae after uptake was reported as percentage of colony forming units after 3 days relative to the total number of cells growing in the absence of DCs exposure. To assess the importance of ROS production in DC killing ability, DPI (10 microM) was added 30 minutes before stimulation and survival of microorganisms was assessed using the same met...

We previously described mRNA electroporation as an efficient gene delivery method to introduce tumor-antigens (Ag) into murine immature dendritic cells (DC).
We have further optimized the protocol and evaluated the capacity of mRNA-electroporated DC as a vaccine for immunotherapy.